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Chapter IV –Enrichment and stabilization of PUFA
Page 100
CHAPTER IV – Enrichment and stabilization of polyunsaturated fatty
acid in recovered lipids from fish visceral waste
n-3 Long-chain polyunsaturated fatty acids (PUFA), especially eicosapentaenoic
acid (EPA, 20:5) and docosahexaenoic acid (DHA, 22:6), have reported to augments
various biochemical changes in degenerative disorders (Russo, 2009; Mantzioris et al.
2000). There have been many reports regarding improving the concentration of PUFAs in
fish oil and marine algae using methods, such as urea inclusion complexation, molecular
distillation, low temperature fractional crystallization, liquid-liquid extraction-fraction,
high performance liquid chromatographic seperation, and salt solubility methods
(Chakraborty and Paulraj, 2007; Guil-Guerrero et al. 2007). However, most of these
methods are not selective for fatty acids and consume a large amount of energy
(Chakraborty et al. 2010). Concentration of n-3 PUFA by enzymatic processes is based
on the use of specific hydrolytic enzymes, like lipases, which catalyze hydrolysis,
ethanolysis or transesterification reaction of triglycerides. Lipase catalyzed concentration
of n-3 PUFA are promising alternative have shown energy saving and are environmental
friendly technology (Shahidi and Wanasundara, 1998). Due to the fatty acid distribution
in the glycerol backbone of triglycerides and the stereospecific activity of certain lipases
(Ando et al. 1996; Gupta et al. 2004), enzymatic hydrolysis is very useful method both in
n-3 PUFA concentration in fish oil and in the production of structured lipids. In this
study, by lipase hydrolysis was attempted to develop a process for simultaneous recovery
and concentration of PUFA from FVW to obtain PUFA enriched fish oil.
One of the major drawbacks in fish oil is their high susceptibility to oxidation,
which results in formation of toxic products (peroxides or volatile compounds) those are
responsible for the non-desirable off-flavours. This is mainly due to their naturally high
content of EPA and DHA. Nevertheless, they are susceptible to oxidation, which is
associated with their rancidity and loss in nutritive value (Frankel et al. 1998). Apart from
high levels of PUFAs, the presence of heme pigments and trace amounts of metallic ions
makes the fish and fish oil prone to lipid oxidation (Hsieh and Kinsella, 1989). To retard
such a quality loss, synthetic antioxidants have been used to decrease lipid oxidation
during the processing and storage of fish and fish products (Boyd et al. 1993).
Chapter IV –Enrichment and stabilization of PUFA
Page 101
Auto-oxidation of PUFA results in free radical generation through reaction
catalyzed by heat, light, trace metals or enzymes (Wang et al. 2011). The free radicals
formed propagate auto-oxidation by reacting with oxygen which results in formation of
hydroperoxides that breakdown to generate other new free radicals (Wang et al. 2011).
Oxidation of lipids can be prevented or minimized during the processing of FVW by like
enzymatic hydrolysis and fermentation. The commonly used antioxidants in food or food
processing are tocopherol, butylated hydroxytoluene (BHT), butylated hydroxyanisole
(BHA) and tertiary butyl hydroquinone (TBHQ). Butylated hydroxyanisole (BHA) is a
phenolic antioxidant that is generally recognized as safe by the FDA but its use is limited
in foods (CFR, 2007; part 172.110). In animal food processing studies, BHA has been
shown to be carcinogenic at high levels (1250 ppm) (Williams e al. 1999). BHT is a
phenolic antioxidant that is generally recognized as safe by the FDA (CFR, 2007; part
172.110). BHT is a fat soluble antioxidant that acts as a synthetic analogue of vitamin E
(Shahidi and Wanasundara, 1992).
The focus of this chapter was to recover lipids with n-3 PUFA-enriched
acylglycerols during fermentation and enzymatic hydrolysis with lipase aided hydrolysis.
Several microbial lipases were used to screen suitable enzyme for n-3 PUFA enrichment
during the fermentation and enzymatic hydrolysis process. Secondly, the effect of various
antioxidants (α-tocopherol, BHT, BHT and TBHQ) in stabilization of PUFA during the
recovery process was also studied. This chapter deals mainly two aspects :
(i) Enrichment of PUFA by lipases during fermentation and enzymatic hydrolysis of
FVW and
(ii) Stabilization of PUFA during fermentation and enzymatic hydrolysis with
antioxidants.
Results
Enrichment of PUFA by lipase hydrolysis during recovery of lipids by lactic acid
fermentation (LAF) and enzymatic hydrolysis (EH)
Enrichment of PUFA in triglyceride (lipids) was carried out by hydrolyzing SFA
and MUFA in glycerol moiety by sn-1,3-specific lipase (Aspergillus niger, Thermomyces
lanuginose, Mucor javanicus) and non specific lipases (Candida rugosa, Candida
cylindrical) (Table 4.1). Abbreviations of these lipases given in the Table 4.1 are used
Chapter IV –Enrichment and stabilization of PUFA
Page 102
hereafter in the text. These lipases were introduced after 24 h of lactic acid fermentation
by P acidilactici NCIM5368 and 60 min after enzymatic hydrolysis using fungal
protease. The protein hydrolysate formed after 24h of lactic acid fermentation or 60 min
of proteolytic hydrolysis acts as an emulsifier for lipase reaction. The recovered oil was
analyzed for triglyceride fatty acid composition and acid value.
Enzyme activity of the lipases
The activity of lipases used for the concentration of PUFA in fish oil recovered
from FVW was assessed for their activity to use an equal enzyme units for the hydrolysis
of triglycerides. Candida cylindrical lipase showed the highest enzyme activity (21740
U/ gm of lipase) among all the lipases used followed by Candida rugosa lipase (10355
U/gm of lipase) while, Aspergillus niger (3391 U/gm), Thermomyces lanuginoca (4460
U/gm of lipase) and Mucor javanicus (3478 U/gm of lipase) showed lower activity
(Table 4.1).
Table 4.1. Properties of lipases used for the hydrolysis of fish oil during the processing
Source of lipase Specificity Activity (Units)
Aspergillus niger (AN) 1-,3->>2- 3391
Candida rugosa (CR) None 10355
Candida cylindrical (CC) None 21740
Thermomyces lanuginoca (TL) 1-,3->>2- 4460
Mucor javanicus (MJ) 1-,3->>2- 3478
These result demonstrate that the non specific lipases used in this study had much
higher activity compared to sn-1,3- specific lipases in hydrolyzing oil recovered from
FVW. All these lipases were used to concentrate PUFA during recovery of oil from
FVW.
Fatty acid composition of FVW-FO triglyceride recovered through solvent extraction,
lactic acid fermentation and enzymatic hydrolysis
Pediococcus acidilactici NCIM5368 and Fungal Protease-P-amano® (P Amano6;
60,000 U/g) were found to be the best lactic acid bacteria and protease for the recovery of
oil from FVW. Therefore they were used for the recovery of oil either by fermentation or
Chapter IV –Enrichment and stabilization of PUFA
Page 103
enzymatic hydrolysis. Fatty acid composition (%) of triglyceride in FVW-FO recovered
through different process is shown in Table 4.2. Irrespective of the process used for the
recovery of oil from FVW, no change in n-3 PUFA profile was observed in triglyceride
of recovered oil. The major fatty acids in the SFA fraction of the oils included palmitic
(16:0) and stearic acid (18:0) whereas palmitoic (14:1) and oleic (C18:1) were the major
MUFA. Linoleic (18:2n-6) and linolenic acid (18:3n-3), arachidonic acid (20:4n-6),
eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3) were the major
PUFA in the oil. Results demonstrate that lactic acid fermentation and enzymatic
hydrolysis of oil does not affect the triglyceride fatty acid composition compared to oil
recovered by solvent extraction.
Table 4.2. Fatty acid composition (%) of triglyceride fraction of FVW-FO recovered by
solvent extraction, lactic acid fermentation and enzymatic hydrolysis
SFA (%) MUFA (%) PUFA (%)
14:0 16:0 18:0 16:1 18:1 18:2n-6 18:3n-3 20:4n-6 20:5n-5 20:5n-5
Fresh 4.3 24.3 7.1 11.2 27.6 8.79 8.22 4.2 2.29 2.72
LAF 4.7 25.2 6.8 10.7 27.1 7.98 8.41 4.5 2.38 2.83
EH 4.1 24.8 6.6 11.4 28.2 8.82 8.78 4.1 2.32 2.70
LAF – Lactic acid fermentation, EH – Enzymatic hydrolysis, SFA – saturated fatty acids,
MUFA – monounsaturated fatty acids, PUFA – polyunsaturated fatty acids; Values are
mean of 5 experiments
Effect of different lipases during fermentation and enzymatic hydrolysis on oil recovery
Oil yield after lactic acid fermentation and enzymatic hydrolysis was calculated as
total oil yield and final oil yield (after removal of FFA). Oil yield on lipase assisted
hydrolysis during LAF of FVW using Pediococcus acidilactici NCIM5368 is shown in
Figure 4.1. There was no significant (p>0.05) difference in total oil yield among different
lipases used during fermentation of FVW using Pediococcus acidilactici NCIM5368.
However, Final oil yield was significantly (p<0.05) lower in case of AN (40.24 %) and
CC (29.8%) lipase followed by TL lipase (32.2%) added before LAF. Similarly, other
lipases assisted hydrolysis also slightly lowered the total oil yield compared to control.
Chapter IV –Enrichment and stabilization of PUFA
Page 104
Acid value increased (2.4 - 5.8 fold) significantly (p<0.05) in the lipase treated oil
compared to control (Figure 4.2). Oil recovered on AN, TL and CC lipase hydrolysis
resulted in significantly (p<0.05) higher acid value compared other lipases (Figure 4.2).
Results on final oil yield and acid value demonstrate that higher degree of hydrolysis of
triglyceride with AN, TL and CC lipase compared to CR and MJ.
0
15
30
45
60
75
90
105
Control AN CR CC TL MJ
Tot
al o
il yi
eld
/ F
inal
oil
yiel
d (
%) Total oil yield Final oil yield
Source of lipases
aa
aa a aa
bb b,c
c c
Figure 4.1. Percent of total and final oil yield on lipase hydrolysis during oil recovery by
lactic acid fermentation of FVW using Pediococcus acidilactici NCIM5368,Values are
mean ± SD (n=5), Values not sharing common alphabet for the same pattern are
significantly different (p<0.05).
Chapter IV –Enrichment and stabilization of PUFA
Page 105
Figure 4.2. Effect of lipases added during lactic acid fermentation of FVW on acid value
(mg KOH required to nutralise FFA) of recovered oil. Values are mean ± SD (n=5),
Values not sharing common alphabet for the same pattern are significantly different
(p<0.05)
In case of enzymatic hydrolysis, similar to lactic acid fermentation there was no
significant (p>0.05) difference in total oil yield when different lipases were used (Figure
4.3). Final oil yield was significantly (p<0.05) lower in comparison to control in lipase
aided enzymatic hydrolysis. The extent of decrease in final oil yield was lower (10.18 –
22.8 %) in enzymatic hydrolysis compared to lactic acid fermentation (21.6 – 40.24 %).
Acid value increased (0.73 - 1.9 fold) significantly (p<0.05) in the lipase treated oil
compared to control. Acid value was higher in case of AN (1.9 fold), TL (1.36 fold) and
CC (1.64 fold) lipases compared to other lipase which followed a similar trend as lactic
acid fermentation (Figure 4.4). The increase in acid value was lower (0.73 - 1.9 fold) in
enzymatic hydrolysis compared to lactic acid fermentation (2.4 - 5.8 fold) which may be
due to lower hydrolysis of triglyceride.
Chapter IV –Enrichment and stabilization of PUFA
Page 106
0
20
40
60
80
100
Control AR CR CC TL MJ
Total oil yield Final oil yield
Source of lipases
Tot
al o
il yi
eld/
Fin
al o
il yi
eld
(%)
a
bb
a aaaa
bb
ba
Figure 4.3. Effect of lipases on total and final oil yield during oil recovery by enzymatic
hydrolysis of FVW using Fungal protease; Values are mean ± SD (n=5),
Values not sharing common alphabet for the same pattern are significantly
different (p<0.05)
Figure 4.4. Effect of lipases added during enzymatic hydrolysis (Fungal proteases) on
acid value (mg of KOH required to neutralize FFA) of recovered oil. Control
– no added lipase; Value are mean ± SD (n=5); Value not sharing common
alphabets for same pattern are significantly different.
Chapter IV –Enrichment and stabilization of PUFA
Page 107
Triglyceride fatty acid composition after hydrolysis
Hydrolysis of FVW triglyceride by lipases caused varying degree of changes in
triglyceride fatty acid composition (Table 4.3). Ideally, enzymatic hydrolysis (lipase)
results in FFA removal that exhibits an increase in EPA and DHA and decrease in SFA
and MUFA concentration. Table 4.3 shows changes in 16:0, 18:0, 16:1, 18:1, 18:3n-3,
20:5n-3 and 22:6n-3 content in the final product obtained after lipase catalyzed
hydrolysis during lactic acid fermentation followed by FFA removal. Among the lipase
used during lactic acid fermentation, hydrolysis of oil with AN lipase resulted in higher
concentration of PUFA (39.7%) compared to control (26.5 %). In specific, hydrolysis
with AN lipase during LAF also resulted in higher concentration of DHA (5.7%)
compared to control (3.1 %). Further, the EPA (46.2%) and ALA (49.2%) content also
increased significantly (p<0.05) in AN hydrolyzed oil compared to control. Among the
lipases used, MM lipase showed the lowest effect on n-3 PUFA concentration. This study
reveals that hydrolysis with sn-1,3 specific lipase AN and TA followed by a non specific
lipase CC resulted in successful concentration of EPA+DHA in FVW-FO.
Effect of lipase on fatty acid composition (%) of lipid recovered on enzymatic
hydrolysis is shown in Table 4.4. Similar to fish oil recovered by LAF, there was increase
in PUFA content which ranged from 3.9 – 25.3% and was higher in AR (25.3 %)
followed by TL (21.4%) and CC (19.1%) lipase treated oil. Extent of concentration of
PUFA was lower in case of lipase added during enzymatic hydrolysis which correlates
with acid value of recovered oil.
Chapter IV –Enrichment and stabilization of PUFA
Page 108
Table 4.3. Fatty acid composition (%) of triglyceride fraction of lipase treated oil
recovered on lactic acid fermentation of FVW.
Fatty acid Control A niger C rugosa C cylindrical T laniginosa M javanicus
Saturated Fatty acids (SFA)
14:0 2.8±0.5a 2.1±0.6a 2.1±0.7a 2.5±0.4a 2.4±0.5a 2.6±0.4a
15:0 1.8±0.3a 1.3±0.3a 1.4±0.2a 1.4±0.3a 1.4±0.2a 1.7±0.3a
16:0 29.7±2.8a 20.6±2.3b 22.6±3.1b 22.0±2.5b 22.7±2.4b 25.7±2.6a.b
17:0 1.7±0.2a 1.4±0.2 a 1.5±0.3a 1.5±0.2a 1.4±0.2a 1.5±0.3a
18:0 5.4±0.6a 3.2±0.3b 4.3±0.5a,c 4.7±0.7a.c 3.8±0.5b,c 4.8±0.5a
ΣSFA 41.4±4.1a 28.6±3.2b 31.9±3.3b 32.1±3.8b 31.7±4.2b 36.3±4.5a,b
Monounsaturated fatty acid (MUFA)
16:1 5.9±0.4a 4.9±0.6a 5.0±0.8a 5.3±0.4a 5.2±0.6a 5.4±0.8a
17:1 0.9±0.1a 0.7±0.1a 0.8±0.2a 0.9±0.1a 0.7±0.1a 0.7±0.2a
18:1n-9 17.8±1.3a 13.4±1.2b 17.2±1.6a 15.6±1.4 a,b 15.7±1.1 a,b 17.0±1.3a
18:1n-7 3.8±0.3a 2.7±0.3 b 3.2±0.3a,b 3.2±0.4a,b 3.6±0.2a 3.5±0.3 a
ΣMUFA 28.4±2.8a 21.7±2.6b 26.2±2.5a,b 25.0±2.6a,b 25.2±2.4a,b 26.6±2.4a,b
Polyunsaturated fatty acids (PUFA)
18:2n-6 10.9±1.3a 14.2±1.2b 12.5±1.1a 12.6±1.0a 12.7±1.4a 11.1±0.3a
18:3n-3 8.7±1.4a 13.0±1.2b 11.0±0.6c 10.7±0.6a,c 10.8±0.4c 10.2±1.3a,c
20:4n-6 1.3±0.4a 2.9±0.6 b 1.7±0.4 a 2.3±0.5 b 2.7±0.4b 1.5±0.3a
20:5n-3 2.6±0.4a 3.8±0.3 b 3.0±0.4 a 3.3±0.4a,b 3.4±0.4a,b 2.8±0.3a
22:6n-3 3.1±0.6a 5.7±0.8 b 3.9±0.7 a,c 4.5±0.6 b,c 4.8±0.5 b,c 3.3±0.4a
ΣPUFA 26.5±2.4a 39.6±2.2b 32.1±2.5c 33.4±2.2 c 34.4±2.9 c 28.9±2.0a
ND – not detected; Values are mean ± SD, Values not sharing common alphabet for the
same row are significantly different (p<0.05).
Chapter IV –Enrichment and stabilization of PUFA
Page 109
Table 4.4. Fatty acid composition (%) of triglyceride fraction of lipase treated oil
recovered by proteolytic hydrolysis of FVW.
Fatty acid Control A niger C rugosa C cylindrical T laniginosa M javanicus
Saturated Fatty acids (SFA)
14:0 3.5±1.2a 2.4±0.9a 3.3±1.0a 2.6±0.8a 2.5±0.9a 2.0±1.1a
15:0 1.5±0.3 a 1.3±0.2a 1.2±0.2a 1.2±0.3a 1.1±0.2a 1.4±0.2a
16:0 29.1±2.2 a 25.6±3.1a 27.5±2.3a 27.0±2.7a 28.9±2.4a 28.7±2.2a
17:0 1.4±0.2 a 1.2±0.2a 1.3±0.1a 1.2±0.3a 1.3±0.2a 1.3±0.3a
18:0 4.8±0.6 a 3.9±0.4a 4.2±0.3a 4.1±0.5a 4.0±0.3a 4.6±0.4a
ΣSFA 40.3±2.9 a 34.4±2.7b 37.5±4.2a,b 36.1±3.6a,b 37.8±3.3a,b 38.0±2.9a,b
Mono – unsaturated fatty acid (MUFA)
16:1 9.0±0.8 a 7.2±0.6b 8.7±0.8 a 8.6±0.7 a 8.2±0.6 a,b 8.7±0.6 a
17:1 1.0±0.1a 0.8±0.1a 0.9±0.1 a 0.8±0.1 a 0.8±0.1 a 1.0±0.2 a
18:1n-9 18.9±1.7 a 16.1±1.4a 17.7±1.5 a 16.6±1.7 a 16.9±1.3 a 18.8±1.6 a
18:1n-7 2.9±0.3 a 2.1±0.3b 2.7±0.3 a,b 2.8±0.2a 2.7±0.2a 2.8±0.3a
ΣMUFA 31.8±2.2 a 26.2±2.1b 30.0±2.4a,b 28.8±2.6 a,b 28.6±2.7 a,b 31.3±2.0 a
Polyunsaturated fatty acids (PUFA)
18:2n-6 10.6±1.1a 12.6±0.9 a 12.2±0.8 a 12.1±1.3 a 12.4±1.2 a 11.1±1.0 a
18:3n-3 8.2±0.7a 10.3±0.8 b 9.3±0.9 a,b 9.6±1.2 a,b 9.7±0.9 a,b 8.4±1.1 a,b
20:4n-6 1.3±0.3a 2.1±0.4b 1.7±0.2a,b 2.0±0.3b 1.9±0.3b,a 1.3±0.2a
20:5n-3 2.6±0.3a 3.3±0.3b 3.1±0.3a,b 3.2±0.4a,b 3.4±0.3b 2.6±0.2a
22:6n-3 3.0±0.3a 3.9±0.4b 3.4±0.4 a,b 3.7±0.5a,b 3.8±0.4b 2.8±0.3a
ΣPUFA 25.7±2.3a 32.2±3.5b. 29.7±2.6a,b 30.6±2.7a,b 31.2±3.4b 26.3±2.6a,b
ND – not detected; Values are mean ± SD, Values not sharing common alphabet for the
same row are significantly different (p<0.05).
Comparing the fermentation and enzymatic hydrolysis methods, lipase aided LAF
has shown to be more effective in concentrating PUFA in recovered fish oil. However, in
both the methods, AN lipase resulted in maximum concentration of PUFA. The decrease
Chapter IV –Enrichment and stabilization of PUFA
Page 110
in SFA and MUFA content on AN lipase treatment during LAF was 30.9 and 23.6 %
respectively whereas during EH was by 14.6 and 17.1% respectively.
Time course study of hydrolysis with Aspergillus niger lipase during lactic acid
fermentation and enzymatic hydrolysis of lipids
As sn-1,3 specific lipase from Aspergillus niger showed better concentration
/enrichment of PUFA compared to other lipases used, effect of hydrolysis time on the
concentration of EPA + DHA in triglyceride was studied. Concentration of EPA and
DHA in triglyceride was estimated during fermentation (at the interval of 4 h till 24 h)
and enzymatic hydrolysis (at the interval of 15 min till 120 min) as shown in Table 4.5.
Table 4.5. Experimental plan for the effect of time on lipase hydrolysis of fish oil during
lactic acid fermentation.
Fermentation
Lipase
Addition
Fermentation + Lipase hydrolysis
Fermentation Time (h) 0 24 28 32 36 40 44 48
Lipase hydrolysis time (h) ___ 0 4 8 12 16 20 24
Figure 4.5. Effect of time (hours) on concentration of EPA and DHA in acylglycerol on
lipase hydrolysis during lactic acid fermentation; Values are mean ± SD, Values
not sharing common alphabet for the same pattern are significantly different
(p<0.05).
Chapter IV –Enrichment and stabilization of PUFA
Page 111
Results show that there was significant increase (p<0.05) in both EPA (2.5 – 3.8
%) and DHA (3.1 – 5.7%) till 16th hour of hydrolysis with no increase afterwards,
suggesting 16 hours of reaction time during LAF may be sufficient enough to obtain
higher levels of EPA and DHA in the final product. There was no change in EPA and
DHA content in control (no lipase) compared to oil recovered by solvent extraction.
After 40 h of LAF (16 h lipase hydrolysis) (Table 4.5), SFA and MUFA content
reduced significantly (p<0.05) whereas there was increase in PUFA content (49.4 %) in
the oil compared to control (Figure 4.6). The concentration of PUFA is may be due to the
hydrolysis of SFA and MUFA in the sn-1,3 position of the triglyceride in the FVW-FO
during LAF and their removal.
0
5
10
15
20
25
30
35
40
45
50
SFA MUFA PUFA n3 PUFA
Control
AR Lipase
%
a
b
a
aa
b
b
b
Figure 4.6. Effect of Aspergillus niger Lipase (16th hour) during lactic acid fermentation
on concentration of SFA (saturated fatty acids), MUFA (monounsaturated fatty acids),
PUFA (polyunsaturated fatty acids) and n-3 PUFA Values are mean ± SD, Values not
sharing common alphabet for the same group of fatty acids are significantly different
(p<0.05).
Effect of time on hydrolysis of fish oil during enzymatic hydrolysis (fungal
protease) was optimized for maximum concentration of PUFA (Table 4.6). In case of
enzymatic hydrolysis, there was consistent and slow increase in EPA and DHA content
till 120th min (180 min of EH) as lipase was added after 60 min of hydrolysis (Figure
4.7). The increase in EPA and DHA during EH on lipase addition was lower compared to
Chapter IV –Enrichment and stabilization of PUFA
Page 112
LAF of FVW. There was significant (p<0.05) change in SFA, MUFA and PUFA content
in the final oil (Figure 4.8) but the decrease was lower compared to oil recovered by
LAF. Results on concentration/enrichment of PUFA suggest that lipase addition during
LAF can be a better approach to improve PUFA levels in recovered fish oil.
Table 4.6. Experimental plan showing the effect of time on lipase hydrolysis of fish oil
during enzymatic hydrolysis (fungal protease).
EH
Lipase
Addition
EH + Lipase hydrolysis
EH Time (min) 0 60 75 90 120 150 180
Lipase hydrolysis time (min) ___ 0 15 30 60 90 120
EH – enzymatic hydrolysis with fungal protease
Figure 4.7. Effect of time (min) on concentration of EPA and DHA in acylglycerol on
lipase hydrolysis during enzymatic hydrolysis (EH).
The comparison of lipase hydrolysis during lactic acid fermentation and enzymatic
hydrolysis of EPA, DHA and PUFA level in the final oil is given in Table 4.7. Results
suggests that lipase hydrolysis during lactic acid fermentation is a better method
compared to proteolytic hydrolysis for the enrichment of PUFA during the recovery of
lipids.
Chapter IV –Enrichment and stabilization of PUFA
Page 113
0
5
10
15
20
25
30
35
40
45
SFA MUFA PUFA n-3 PUFA
Control AR Lipase
%
a
ba
a
ab
b
b
Figure 4.8. Effect of Aspergillus niger lipase hydrolysis during enzymatic hydrolysis
(Fungal protease) on concentration of SFA (saturated fatty acids), MUFA
(monounsaturated fatty acids), PUFA (polyunsaturated fatty acids) and n-3 PUFA;
Values are mean ± SD, Values not sharing common alphabet for the same group of fatty
acids are significantly different (p<0.05).
Table 4.7. Comparison of PUFA enrichment during lactic acid fermentation and
enzymatic hydrolysis
Process EPA DHA PUFA
Solvent extracted 2.5±0.2a 3.1±0.2a 26.3±2.4a
LAF 2.7±0.2a 3.0±0.4a 26.4±3.1a
EH 2.6±0.2a 3.1±0.2a 25.7±2.6a
LAF + AN lipase 3.9±0.3b 5.8±0.4b 39.7±4.1b
EH + AN lipase 3.3±0.3c 3.9±0.4c 32.2±2.3c
LAF – lactic acid fermentation, EH – enzymatic hydrolysis, PUFA – polyunsaturated
fatty acid, AN – Aspergilus niger, Values are mean ± SD, Values not sharing common
alphabet for the same column are significantly different (p<0.05)
Chapter IV –Enrichment and stabilization of PUFA
Page 114
Effect of antioxidants on oxidation of FO recovered during lactic acid fermentation
Different antioxidants (α-tocopherol, BHT, BHA, TBHQ at 100ppm) were to
prevent the auto-oxidation of PUFA during the recovery of FVW-FO. The data presented
in Figure 4.9 reflects that antioxidants had a significant (p<0.05) effect on the formation
of oxidation products in the sample. Results show a lower PV value in antioxidant added
FVW compared to control FVW (no antioxidant added). The PV value (meq of O2/kg of
oil) of control fermented sample was 37.8 which reduced significantly (p<0.05) in the
BHT (40.9%), BHA (46.4 %) TBHQ (71.5 %) and tocopherol (63.1 %) added samples.
The lowest PV (meq of O2/kg of oil) value was found in TBHQ (10.8) added FVW
followed by α-tocopherol (13.5) added indicating superior antioxidant properties of
antioxidant used in that order.
Figure 4.9. Effect of antioxidants on peroxide value (PV) and acid value of oil recovered
on fermentation of FVW with Pediococcus acidilactici NCIM5368. BHT - butylated
hydroxytoluene, BHA - butylated hydroxyanisole, TBHQ tertiary butyl hydroquinone
(TBHQ), Control – without antioxidant, Acid value – expressed as mg KOH required to
nutralize FFA; Values are mean ± SD (n=5) (n=5), Values not sharing common alphabet
for the same pattern are significantly different (p<0.05)
In case of acid value, there was no significant (p>0.05) difference among lipids
recovered on addition of antioxidants to homogenized FVW (Figure 4.9). Fatty acid
composition (%) of lipid recovered on fermentation with added antioxidant did not show
Chapter IV –Enrichment and stabilization of PUFA
Page 115
any change in fatty acid composition (Table 4.8). In particular, concentration of PUFA
remained unchanged irrespective of the antioxidant added. As TBHQ and α-tocopherol
were the two potent antioxidant found to reduce the peroxide value without affecting the
acid value and fatty acid composition, hence they were used for further study.
Table 4.8. Effect of antioxidants on fatty acid composition of fish oil recovered by lactic
acid fermentation of fish visceral waste.
Fatty acids Tocopherol BHT BHA TBHQ Fresh Control
14:0 1.8±0.2 a 1.7±0.2 a 1.8±0.2 a 1.8±0.3 a 1.9±0.3 a 1.7±0.2 a
15:0 0.7±0.1 a 0.7±0.2 a 0.7±0.1 a 0.7±0.1 a 0.8±0.2 a 0.7±0.1 a
16:0 26.5±2.8 a 26.3±3.0 a 26.5±3.6a 28.1±2.5 a 27.2±2.9a 25.8±3.2 a
17:0 1.4±0.3 a 1.4±0.2 a 1.4±0.2 a 1.4±0.3 a 1.3±0.3 a 1.1±0.2 a
18:0 6.5±0.4 a 6.5±1.1 a 6.5±0.6 a 6.2±0.9 a 6.4±0.4 a 6.2±0.7 a
16:1 5.4±0.7 a 5.6±0.5 a 5.8±0.8 a 5.7±0.7 a 5.1±0.9 a 5.4±0.4 a
17:1 0.7±0.2 a 0.6±0.2 a 0.7±0.2 a 0.7±0.1 a 0.8±0.1 a 0.8±0.2 a
18:1n-9 25.0±3.4 a 25.0±2.7 a 24.8±2.5a 25.0±2.8 a 24.8±3.2a 24.3±3.0 a
18:1n-7 2.3±0.3 a 2.2±0.2 a 2.3±0.3 a 1.8±0.3 a 1.9±0.4 a 2.1±0.3 a
18:2n-6 10.2±0.9 a 9.1±1.1 a 9.2±0.8 a 8.6±1.6 a 8.8±1.4 a 9.4±0.8 a
18:3n-3 8.8±1.0 a 8.6±0.7 a 8.0±0.6 a 8.5±0.8 a 8.4±1.2 a 7.8±0.9 a
20:4n-6 1.1±0.3 a 1.2±0.3 a 1.6±0.4 a 1.2±0.2 a 1.1±0.2 a 1.6±0.4 a
20:5n-3 2.4±0.2 a 2.4±0.2 a 2.3±0.3 a 2.4±0.3 a 2.4±0.2 a 2.2±0.3a
22:6n-3 2.7±0.3 a 2.6±0.3 a 2.6±0.2 a 2.7±0.2 a 2.6±0.3 a 2.4±0.2a
BHT - butylated hydroxytoluene, BHA - butylated hydroxyanisole, TBHQ tertiary butyl
hydroquinone (TBHQ), Fresh – oil recovered by solvent extraction, control – LAF
without antioxidant; Values are mean ± SD (n=5), Values not sharing common alphabet
for the same pattern are significantly different (p<0.05)
As TBHQ and α-tocopherol were the better antioxidant, experiment for
optimization of their minimum concentration to prevent oxidation of PUFA was carried
out. LAF was carried out at different concentration (50, 100, 150 and 200ppm) of
selected antioxidant. Among the antioxidant used, TBHQ (68.5 %) and α-tocopherol
Chapter IV –Enrichment and stabilization of PUFA
Page 116
(55.2%) showed significant (p<0.05) reduction in PV at lowest concentration of 50 ppm.
TBHQ and α-tocopherol showed the maximum reduction of 100% at 100 ppm and 150
ppm respectively, beyond which there was no change in peroxide value. The values were
similar to the peroxide values of the freshly extracted fish oil by solvent extraction
(Figure 4.10).
0
5
10
15
20
25
30
35
40
50 100 150 200 Fresh Control
TBHQ α-Tocopherol
Concentration in ppm
mg
of O
2/kg
oil
a cb
bb
a
bb
dc
cc
Figure 4.10. Effect of different concentration of TBHQ (tertiary butyl hydroquinone) and
α-tocopherol on peroxide value (meq of O2/kg) of oil recovered by lactic
acid fermentation. Fresh – oil recovered by solvent extraction, control –
without antioxidant; Values are mean ± SD (n=5), Values not sharing
common alphabet for the same pattern are significantly different (p<0.05)
Studies on the effect of antioxidants added to FVW before lactic acid fermentation
by Pediococcus acidilactici NCIM5368 suggests that addition of 100 ppm of TBHQ or
150 ppm of α-tocopherol to FVW can prevent the oxidation of PUFA during the
fermentation process of FVW.
Chapter IV –Enrichment and stabilization of PUFA
Page 117
Effect of antioxidants on oxidation of FO recovered during enzymatic hydrolysis
The effect of TBHQ, BHT, BHA and α-tocopherol added to homogenized FVW
before initiation of enzymatic hydrolysis showed a significant (p<0.05) reduction in the
formation of oxidation product in the recovered fish oil. The PV value was lower in oil
recovered from antioxidant added FVW (before hydrolysed with Fungal protease)
compared to control (no antioxidant). The PV value of control oil was 37.5 meq O2/kg of
which reduced significantly (p>0.05) in tocopherol (66.7%), TBHQ (75.7%), BHT
(50.5%) and BHA (58.5 %) added groups (Figure 4.11). The lowest PV value (meq O2/kg
of oil) in fish oil stabilized with TBHQ (9.1) and α-tocopherol (12.5) at 100ppm
concentration demonstrating that these antioxidants are the best for the stabilization of
FVW-FO.
0
5
10
15
20
25
30
35
40
45
α-Tocopherol BHT BHA TBHQ Fresh Control
Acid value Peroxide value
Antioxidants
Aci
d /
Per
oxid
e va
lue
a
b
a aaaaa
c
d d
e
Figure 4.11. Effect of antioxidants on peroxide value (meq of O2/kg of oil) and acid
value (mg KOH/ kg of oil) of oil recovered on enzymatic hydrolysis of FVW with Fungal
proteases. BHT - butylated hydroxytoluene, BHA - butylated hydroxyanisole, TBHQ -
tertiary butyl hydroquinone, Fresh – oil recovered by solvent extraction, control –without
antioxidant. ; Values are mean ± SD (n=5), Values not sharing common alphabet for the
same pattern are significantly different (p<0.05)
There was no significant (p>0.05) change in the acid value of fish oil recovered
from FVW by enzymatic hydrolysis with added antioxidants. However, acid value of FO-
Chapter IV –Enrichment and stabilization of PUFA
Page 118
EH was non-significantly higher in case of BHT (11.9 %) and BHA (10.8 %) added
FVW. Effect of α-tocopherol, TBHQ, BHT and BHA on fatty acid composition of FVW-
FO is shown in Table 4.9. Addition of antioxidants to FVW before enzymatic hydrolysis
did not change the fatty acid composition of the recovered oil compared to that of
recovered by solvent extraction. In particular, concentration of n-3 PUFA (EPA, DHA
and ALA) remained unchanged irrespective of antioxidant added (Table 4.8).
Table 4.9. Effect of antioxidants on fatty acid composition (%) of fish oil recovered by
enzymatic hydrolysis of fish visceral waste
Fatty acids α-Tocopherol BHT BHA TBHQ Fresh Control
14:0 1.8±0.2a 1.7±0.1a 1.8±0.2a 1.8±0.2a 1.9±0.2a 1.7±0.2a
15:0 0.7±0.2a 0.7±0.8a 0.7±0.1a 0.7±0.1a 0.8±0.2a 0.7±0.1 a
16:0 26.5±2.8a 26.3±2.5a 26.5±2.6a 28.1±3.1a 27.2±2.4a 25.8±3.0a
17:0 1.4±0.3a 1.3±0.2a 1.4±0.2a 1.4±0.3a 1.3±0.2a 1.1±0.2a
18:0 6.5±0.4a 6.6±0.7a 6.5±1.1a 6.2±0.9a 6.4±0.8a 6.2±0.5a
16:1 5.4±0.5a 5.6±0.8a 5.8±0.6a 5.7±0.7a 5.1±0.4a 5.4±0.6a
17:1 0.7±0.1a 0.6±0.2a 0.7±0.2a 0.7±0.1a 0.8±0.1a 0.8±0.1a
18:1n-9 25.0±3.2a 25.0±2.8a 24.8±2.9a 25.0±2.7a 24.8±3.1a 24.3±2.6a
18:1n-7 2.3±0.2a 2.2±0.2a 2.3±0.3a 1.6±0.3a 1.9±0.2a 2.1±0.2a
18:2n-6 10.2±1.6a 9.1±0.7a 9.2±1.2a 8.6±1.0a 8.8±0.7a 9.4±0.5a
18:3n-3 8.2±0.5a 8.3±0.9a 8.0±0.4a 8.4±0.6a 8.2±0.7a 7.9±0.7a
20:4n-6 2.4±0.2a 2.5±0.3a 2.6±0.3a 2.4±0.3a 2.5±0.3a 2.6±0.2a
20:5n-3 2.3±0.2a 2.2±0.2a 2.2±0.1a 2.3±0.3a 2.3±0.2a 2.1±0.2a
22:6n-3 2.8±0.2a 2.7±0.2a 2.6±0.3a 2.8±0.3a 2.8±0.2a 2.6±0.2a
BHT - butylated hydroxytoluene, BHA - butylated hydroxyanisole, TBHQ tertiary butyl
hydroquinone, Control – without antioxidant. Fresh – oil recovered by solvent extraction,
control – enzymatic hydrolysis without antioxidant; Values are mean ± SD (n=5), Values
not sharing common alphabet for the same pattern are significantly different (p<0.05)
TBHQ and α-tocopherol were found to be the better antioxidants in preventing
oxidation of PUFA during the recovery of FVW-FO. Hence, they were chosen for further
studies to find out their concentration dependent effect on oxidation. EH of FVW was
Chapter IV –Enrichment and stabilization of PUFA
Page 119
carried out with 50, 100, 150 and 200ppm of the selected antioxidant. PV of control oil
recovered by enzymatic hydrolysis of FVW was 36.5 meq of O2/kg of oil which reduced
significantly (p<0.05) on addition of TBHQ (50 ppm) and α-tocopherol (50 ppm) (Figure
4.12). Concentration of TBHQ and α-tocopherol required to mentain the PV (reduce the
auto-oxidation) similar to fresh (extracted by solvent) in 100 ppm and 150 ppm
respectively.
0
5
10
15
20
25
30
35
40
45
50 100 150 200 Fresh Control
TBHQ α-Tocopherol
Concentration in ppm
meq
of
O2/
kg o
f oi
l
aa
b
dc
bbb
b ccc
Figure 4.12. Effect of concentration of TBHQ and α-tocopherol on peroxide value (meq
of O2/kg of oil) of oil recovered by enzymatic hydrolysis. TBHQ - tertiary
butyl hydroquinone. Fresh – oil recovered by solvent extraction, control –
LAF without antioxidant; Values are mean ± SD (n=5), Values not sharing
common alphabet for the same pattern are significantly different (p<0.05)
In general effect of TBHQ on PV and acid value during LAF and EH of FVW
was found to be similar. The results further demonstrate that TBHQ is a better
antioxidant compared to other antioxidant used. The results conclude that 100 ppm of
TBHQ or 150 ppm of α-tocopherol can be recommended to reduce the oxidation of
recovered fish oil during LAF or EH of FVW. These antioxidants can be added to
homogenized FVW at the recommended concentration to stabilize PUFA during the
recovery of fish oil from FVW.
Chapter IV –Enrichment and stabilization of PUFA
Page 120
Discussion
Enrichment/concentration of PUFA using lipases in recovered oil
In the previous chapter standardization of lactic acid fermentation and enzymatic
hydrolysis for the recovery of lipids from FVW has been carried out. Fatty acid
composition of FO-LAF and FO-EH showed equal distribution of SFA, MUFA and
PUFA. PUFA concentrates in triglycerides, devoid of more saturated fatty acids, are
much better than original oil themselves because they allowed daily intake of total lipid
to be as low as possible.
Lipase aided hydrolysis during recovery of FVW-FO caused changes in acid value
and fatty acid composition at varying levels. Higher concentration of PUFA in
triglyceride was observed in case of lipase aided hydrolysis during lactic acid
fermentation compared to proteolytic enzymatic hydrolysis. Lipase catalyzed hydrolysis,
especially with AN and TL sn-1,3-specific lipases resulted in successful concentration of
EPA and DHA with different efficiencies. Ideally, enzymatic hydrolysis using lipases
followed by removal FFA increases the EPA and DHA concentrations by reducing SFA
and MUFA. Aspergillus niger sn-1,3-specific lipase used in the study was significantly
higher compared to other lipases in concentrating PUFA during the recovery process.
Okada and Morrissey (2007) have reported that non-specific lipases was better than sn-1,
3-specific lipase in concentrating PUFA in sardine oil. In case of fish oil, the second
position of the glycerol moiety is usually more enriched with n-3 PUFAs (Bornscheuer,
2000), although this may vary depending on species (Gamez-Meza et al. 1999). The
results on concentration of PUFA by sn-1,3 specific lipase also highlights the presence of
n-3 PUFA on the sn-2 position in FVW-FO.
Concentration of PUFA was also observed in the non-specific lipase CC in this
study. This indicates the ability of CC lipases to discriminate SFAs and MUFAs from n-3
PUFA in FO-FVW, most likely due to the reduced steric hindrance observed with SFAs
and MUFAs when linked to a glycerol backbone (Gamez-Meza et al. 2003). The
molecular conformation of cis carbon–carbon double bonds in PUFAs, particularly EPA
and DHA, causes steric hindrance and subsequent bending of the fatty acid chains,
bringing the terminal methyl groups very close to the ester bonds (Chakraborty et al.
2010). Because of this steric hindrance effect, enzymatic active sites cannot reach the
Chapter IV –Enrichment and stabilization of PUFA
Page 121
ester-linkages of these fatty acids with their glycerol backbones, thereby protecting EPA
and DHA from lipase-catalyzed hydrolysis. However, this does not occur with the
relatively straight chains of SFAs and MUFAs, and therefore hydrolysis is not hindered
(Shahidi and Wanasundara, 1998; Carvalho et al. 2002). Also, it has been suggested that
TGs without EPA and DHA are hydrolyzed in the first phase, and TGs with EPA and
DHA are hydrolyzed later, indicating that the lipase recognizes the whole molecular
structure, not only its ester bonds (Hoshino et al. 1990).
Lipase catalyzed hydrolysis was demonstrated to be a feasible method for
concentration of n-3 PUFAs during lactic acid fermentation and enzymatic hydrolysis.
Use of lipase to produce n-3 PUFA concentrates has an advantage over traditional
methods such as chromatographic separation, molecular distillation etc. of concentration
because such procedures involves extreme pH and high temperature which may affect the
quality of oil. Therefore, the mild conditions using enzymatic hydrolysis provide a
promising alternative that could also save energy and increase product selectivity. In
addition, the enzymatic hydrolysis method produces n-3 fatty acids in the glycerol form,
which is considered nutritionally favorable.
Stabilization of PUFA during fermentation and enzymatic hydrolysis
Due to the presence of multiple double bonds in PUFAs, they are highly susceptible
to oxidation and the oxidation products can have adverse health effects to the consumer
due to their cytotoxic and genotoxic effects (Esterbauer, 1993). Peroxide value (PV) is a
measure of the extent of oxidation of a lipid system. This value indicates the quantity of
oxidized substances, normally hydroperoxides, which liberate iodine from potassium
iodide under specified conditions (Rogers et al. 2001; Yanishlieva and Marinova, 2001).
Fish oil during lactic acid fermentation and enzymatic hydrolysis may decompose readily
which is measured by an increase in PV. In our present study, different antioxidants were
assessed find to find out their ability to protect PUFA from oxidation during the recovery
of FVW-FO. Results have shown that 100 ppm of TBHQ and 150 ppm of α-tocopherol
prevents auto-oxidation of fish oil during recovery of oil by lactic acid fermentation and
enzymatic hydrolysis.
Antioxidants function by inhibiting or interrupting the free radical mechanism of
lipid auto-oxidation. Antioxidants or phenolic substances function as free radical
Chapter IV –Enrichment and stabilization of PUFA
Page 122
acceptors, thereby terminating oxidation at the initial step and also scavenging radicals
formed later, in the oxidation process (Wang et al.2011). The antioxidant and free radical
complex is stable and does not split into other compounds that provide off-flavor and
odors, nor does it propagate further oxidation of the lipid (O’Brien, 1998). In the present
study, TBHQ and α-tocopherol were found to be the best antioxidant compared to other
in reducing peroxide value. Previous studied by Haung et al (1994) with corn oil and by
Kulas and Ackman (2001) on fish oil showed 100 ppm α-tocopherol as a concentration
for maximal antioxidant activity in those oil.
It is concluded that TBHQ (100ppm) and α-tocopherol (150ppm) minimizes the
oxidation of fish oil during fermentation and enzymatic hydrolysis. These antioxidants
can be used to homogenized FVW before lactic acid fermentation/enzymatic hydrolysis
to recover good quality (unoxidized) lipids. In case of enrichment/ concentration of
PUFA by lipase treatment 1,3 specific AN lipase was the best among the lipase used in
our study for both LAF and EH.
Recommended